BR112021015654A2 - COMPOSITIONS FOR CRYOPRESERVATION OF A BIOLOGICAL MATERIAL - Google Patents

Abstract

compositions for cryopreservation of biological material. The present application discloses a liquid composition for cryopreservation of a biological material comprising an aprotic polar solvent, a monohydric or polyhydric alcohol, an unbranched polysaccharide, a branched polysaccharide; and polyvinyl alcohol. furthermore, uses of said liquid composition for cryopreservation of a biological material and methods for preserving a biological material using said liquid composition are provided.

Description

Descriptive Report of the Patent of Invention for: “COMPOSITIONS FOR CRYOPRESERVATION OF A BIOLOGICAL MATERIAL”

FIELD

[001] The present invention relates to liquid compositions for cryopreservation of a biological material, methods employing these compositions and their uses. The present invention is particularly useful for the cryopreservation of human cells, gametes and embryos.

FUNDAMENTALS

[002] Cryopreservation is among the most powerful and efficient tools to indefinitely preserve the genetics of economically, therapeutically or scientifically important cells, embryos or gametes. This also applies to assisted human reproduction (ART) technologies. As water is the main component of the cell, its solidification must be tightly controlled during cooling and subsequent heating to prevent intracellular formation of ice crystals. This would have destructive effects on organelles and all membrane systems, resulting in deleterious cellular changes and death.

[003] Two main groups of methods are currently in use for the cryopreservation of living biological material: slow freezing (SLF) and vitrification (VIT). VIT procedures were introduced as an alternative to SLF to reduce the likelihood of intracellular ice crystal formation (Rall and Fahy 1985).

[004] VIT has been shown to be more efficient than SLF in human TRA, particularly for cryopreservation of metaphase II (MII) oocytes (Kuwayama et al. 2005) and embryos at various stages of development (Rienzi et al. 2017; Vanderzwalmen et al. . 2012), now standing as the gold standard for cryopreservation in ART.

[005] This is equally true for animal and particularly murine embryos, where VIT has been shown to better preserve chromatin integrity and bioenergy status (Somoskoi et al. 2015), inducing lower intracellular ingress of cryoprotectants (CPs) (Vanderzwalmen et al.

2013) and ultimately produce better embryo survival and development than SLF (Zander-Fox et al.

2013).

[006] However, current VIT protocols suffer from methodological complexity, multiplicity of protocols, and frequent use of chemically undefined components, often originating from human or animal fluids.

As a result, component stability, biological safety, reproducibility and process standardization are compromised.

[007] In view of this, there is a need for additional and/or improved compositions for vitrification -

and also slow freezing - of cells with high efficiency, biological safety and reproducibility.

SUMMARY

[008] The present inventors have developed a unique chemically defined liquid composition for cryopreservation of a biological material. The inventors have found that the specific combination of reagents allows for a synergistic effect of these reagents, resulting in optimal characteristics for use in cryopreservation. More particularly, the use of the compositions of the invention guarantees better survival and hatching rate than known cryopreservation solutions.

[009] This liquid composition allows efficient, biologically safe and reproducible cryopreservation (e.g. vitrification) of various types of biological material, encompassing - but not limited to - stem cells, embryos and gametes, by various types of cryopreservation procedures (e.g. slow freezing, one-step glazing, multi-step glazing).

[0010] Accordingly, a first aspect provides a liquid composition for cryopreservation of a biological material comprising a polar aprotic solvent, a monohydric or polyhydric alcohol and a polyvinyl alcohol.

In particular modalities, the composition comprises:

- a polar aprotic solvent selected from dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) - a monohydric or polyhydric alcohol selected from ethylene glycol, propylene glycol, glycerol, erythritol, sorbitol, mannitol, xylitol, volemitol, methanol, ethanol, isopropanol, butanol and pentanol; - an unbranched polysaccharide selected from the group consisting of sucrose, trehalose, lactulose, melibiose, lactobionate, raffinose and cellulose, preferably sucrose; - a branched polysaccharide selected from the group consisting of dextran, pectin, Ficoll and starch, preferably dextran; and - polyvinyl alcohol.

[0011] In particular embodiments, the liquid composition for cryopreservation of a biological material comprises DMSO, ethylene glycol, sucrose, dextran and polyvinyl alcohol.

[0012] In particular embodiments, the liquid composition for cryopreservation of a biological material comprises substantially equal amounts of DMSO and ethylene glycol and a concentration of sucrose that is at least 8 times that of dextran.

[0013] In particular embodiments, the liquid composition for cryopreservation of a biological material comprises: - at least 0.8% (v/v) of a polar aprotic solvent; - at least 0.8% (v/v) of a monohydric or polyhydric alcohol; - at least 0.5% (w/v) of unbranched polysaccharide; - at least 0.037% (w/v) of a branched polysaccharide; - at least 0.015% (w/v) of polyvinyl alcohol; and - up to 100% (v/v) of a diluent, preferably pH 7.2 to 7.4.

[0014] In a preferred embodiment, the liquid composition for cryopreservation of a biological material comprises: - at least 0.8% (v/v) of a polar aprotic solvent; - at least 0.8% (v/v) of a monohydric or polyhydric alcohol; - at least 0.5% (w/v) of unbranched polysaccharide; - at least 0.037% (w/v) of dextran; - at least 0.015% (w/v) of polyvinyl alcohol; and - up to 100% (v/v) of a diluent, preferably with pH 7.2 to 7.4.

[0015] In particular embodiments, the liquid composition for cryopreservation of a biological material comprises: - 13.0 to 23.0% (v/v), preferably 17.7% (v/v) of a polar solvent aprotic; - 13.0 to 23.0% (v/v), preferably 17.7% (v/v), of a monohydric or polyhydric alcohol; - 9.0 to 31.0% (w/v), preferably 22.7% (w/v), of an unbranched polysaccharide; - 0.60 to 22.0% (w/v), preferably 2.64% (w/v), of a branched polysaccharide; - 0.30 to 4.0% (w/v), preferably 0.88% (w/v), of polyvinyl alcohol; and/or - up to 100% (v/v) of a diluent, preferably with pH 7.2 to 7.4; or a dilution of said liquid composition in said diluent.

[0016] In a preferred embodiment, the liquid composition for cryopreservation of a biological material comprises: - 13.0 to 23.0% (v/v), preferably 17.7% (v/v) of a solvent aprotic polar; - 13.0 to 23.0% (v/v), preferably 17.7% (v/v), of a monohydric or polyhydric alcohol; - 9.0 to 31.0% (w/v), preferably 22.7% (w/v), of an unbranched polysaccharide; - 0.60 to 22.0% (w/v), preferably 2.64%

(w/v), dextran; - 0.30 to 4.0% (w/v), preferably 0.88% (w/v), of polyvinyl alcohol; and/or - up to 100% (v/v) of a diluent, preferably with pH 7.2 to 7.4; or a dilution of said liquid composition in said diluent.

[0017] In particular embodiments, the liquid composition for cryopreservation of a biological material comprises at least 15% (v/v) of a polar aprotic solvent, at least 15% (v/v) of a monohydric or polyhydric alcohol, at least 15% (w/v) of an unbranched polysaccharide, at least 2% (w/v) of a branched polysaccharide and about 0.9% (w/v) of polyvinyl alcohol.

[0018] In a preferred embodiment, the liquid composition for cryopreservation of a biological material comprises at least 15% (v/v) of a polar aprotic solvent, at least 15% (v/v) of a monohydric or polyhydric alcohol, at least at least 15% (w/v) of an unbranched polysaccharide, at least 2% (w/v) of dextran and about 0.9% (w/v) of polyvinyl alcohol.

[0019] In particular embodiments, said diluent is balanced saline, preferably phosphate buffered saline (PBS).

[0020] In particular embodiments, dextran comprises one or more of Dextran 1, Dextran 40 and Dextran

70.

[0021] In particular modalities, the liquid composition for cryopreservation of a biological material comprises: - 17.7% (v/v) of DMSO, - 17.7% (v/v) of ethylene glycol, - 22.7% ( w/v) of sucrose, - 0.88% (w/v) of dextran 1 (D1) - 0.88% (w/v) of dextran 40 (D40) - 0.88% (w/v) of dextran 70 (D70); and - 0.88% (w/v) of polyvinyl alcohol.

[0022] In particular embodiments, the liquid composition does not comprise proteins, polypeptides or peptides.

[0023] Another aspect provides the use of the liquid composition, as taught herein, for the cryopreservation of a biological material.

[0024] In particular embodiments, cryopreservation of biological material is performed by a freezing procedure, a multi-step vitrification procedure or a one-step vitrification procedure.

[0025] Another aspect provides a method for preserving a biological material comprising the steps of - providing a biological material;

- contacting the biological material with the liquid composition as taught in this document; - cooling and/or heating the biological material in the liquid composition, as taught herein. In particular embodiments, the method further comprises contacting the biological material with at least one dilution of the liquid composition as taught herein, prior to contacting the biological material with said composition.

[0026] In particular modalities, said cooling comprises the freezing of the biological material or the vitrification of the biological material.

[0027] In particular embodiments, said method comprises vitrification in one or several stages of the biological material.

[0028] In particular modalities, the biological material is selected from the group of stem cells, gametes and embryos.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 represents the viability and confluence of mesenchymal stem cells isolated from equine cadavers (EC-MSCs) after one-step vitrification using the liquid composition (also referred to herein as cryopreservation solution (CDCS)) as taught in this document. A: Cell viability of EC-MSCs 24h post-warming (vitrified samples: mean = 87.9%) or post-warming

passage (unvitrified samples: mean 94.5%) determined by trypan blue exclusion assay. Data represent the mean + standard deviation (SD) of 15 and 20 samples (unvitrified and vitrified, respectively) from 5 independent experiments. (Mann-Whitney test: P < 0.001); B to F: Comparison of the confluence of EC-MSCs, reflecting the number of viable cells as well as their ability to proliferate, between vitrified and non-vitrified samples in 5 independent experiments. Data points represent mean ± SD; the number of samples in each group is indicated in square brackets.

[0030] Figure 2 represents the morphology of vitrified and non-vitrified EC-MSCs after 3 days of culture.

Left side: unglazed controls; right side: EC-MSCs vitrified using CDCS composition as taught in this document following the one-step method.

Scale bar: 50 m.

[0031] Figure 3 depicts the viability and confluence of EC-MSCs after slow freezing using 2x (ie 50% dilution) and 4x (ie 25% dilution) diluted CDCS stock solution. A: Cell viability of EC-MSCs 24h post-thawing (cryopreserved samples) or post-passage (non-cryopreserved samples) (mean 93.7%, 86.8%, 90.6% and 93% for non-cryopreserved control, reference method (Ref), 50% of CDCS samples and 25%

of CDCS, respectively) determined by the trypan blue exclusion assay. Data represent the mean + SD of 5 samples (Mann-Whitney test: **P < 0.01; ns: not significant); B: Comparison of EC-MSCs confluence, reflecting the number of viable cells as well as their ability to proliferate, between the reference freezing method (Ref), 2x and 4x diluted CDCS (50% and 25%, respectively) and non-cryopreserved samples. Data points represent mean ± SD; the number of samples in each group is indicated in square brackets.

[0032] Figure 4 represents the viability and confluence of human-induced pluripotent stem cells (hiPSCs) after slow freezing using CDCS. A: Cell viability of hiPSCs 24h after thawing (cryopreserved samples) or post-passage (non-cryopreserved samples) determined by trypan blue exclusion assay. Data represent the mean + SD of 6 to 7 samples from 2 independent experiments; (Mann-Whitney test: *P < 0.05, **P < 0.01) B: Representative example illustrating the comparison of confluence of hiPSCs between frozen and non-frozen samples. Data points represent mean ± SD; the number of samples in each group is indicated in square brackets.

DESCRIPTION

[0033] As used in this document, the singular forms "a", "an", and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0034] The terms "comprising", "comprises" and "comprised of", as used in this document, are synonymous with "including", "includes" or "containing", "contains" and are inclusive or open and do not exclude members , additional or not mentioned elements or method steps. The terms also encompass "consisting of" and "consisting essentially of". The term "consisting of", as used herein, when referring to a composition, means that no other ingredients are present in said composition than those recited later. The term "consisting essentially of", when referring to a composition, allows for the presence of traces of reagents, but normally at a concentration of less than 0.1% w/v.

[0035] Citation of numeric ranges by endpoints includes all numbers and fractions included in the respective ranges, as well as the quoted endpoints.

[0036] The terms "about" or "approximately", as used herein, when referring to a measurable value, such as a parameter, an amount, a time duration, and the like, are intended to encompass variations to and from of the specified value, as variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1% or less, and even more preferably +/- 0.1% or less of and from the specified value, to the extent such variations are appropriate for carrying out the disclosed invention. It should be understood that the value to which the modifier "about" refers is also specifically, and preferably, disclosed.

[0037] Whereas the terms "one or more" or "at least one", such as one or more members or at least one member of a group of members, are clear per se, by way of further exemplification the term encompasses, inter alia, a reference to any of said members, or to any two or more of said members, such as, for example, any  3,  4, 5,  6 or  7 etc. of said members, and even all said members. In another example, "one or more" or "at least one" can refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0038] Discussion of the background of the invention in this document is included to explain the context of the invention.

This should not be taken as an admission that any material referred to was published, known or common general knowledge in any country on the priority date of any of the claims.

[0039] Unless defined otherwise, all terms used in the disclosure of the invention, including technical and scientific terms, have the meaning as commonly understood by a person skilled in the art to which this invention pertains. By way of further guidance, definitions of terms are included to better recognize the teachings of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation shall apply throughout this specification, i.e. also in the context of other aspects or embodiments of the invention, unless defined accordingly. other way.

[0040] In the following passages, different aspects or embodiments of the invention are defined in more detail.

Each aspect or modality so defined may be combined with any other aspect or modality, unless clearly stated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0041] Reference throughout this specification to "an embodiment" means that a particular feature, structure, or feature described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases "in one modality" in various places throughout this descriptive report does not necessarily refer to the same modality, but it may. Furthermore, particular features, structures or features may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described in this document include some, but not other features included in other embodiments, combinations of features from different embodiments should be within the scope of the invention and form different embodiments, as would be understood by those skilled in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0042] The present inventors have developed a unique chemically defined liquid composition for cryopreservation of a biological material. The inventors have found that the specific combination of reagents allows for a synergistic effect of these reagents, resulting in optimal characteristics for use in cryopreservation.

[0043] As detailed herein, the compositions of the present invention comprise, consist essentially of or consist of a polar aprotic solvent (e.g. dimethyl sulfoxide (DMSO)), a monohydric or polyhydric alcohol (e.g. ethylene glycol), a non (e.g. sucrose) and a branched polysaccharide (e.g. dextran) and polyvinyl alcohol (PVA) ((the liquid composition is also referred to herein as a cryopreservation solution (CDCS)). biologically safe and reproducible (e.g. vitrification) of various types of biological material, encompassing - but not limited to - stem cells, embryos and gametes, by various types of cryopreservation procedures (e.g. slow freezing, vitrification in a step, multi-step vitrification).

More particularly, the liquid composition allows for efficient cryopreservation (e.g., vitrification) of biological material generally with viability, proliferation, and morphological parameters similar to non-cryopreserved control biological material, or similar or better than biological material cryopreserved with a solution of reference control cryopreservation.

[0044] All ingredients of the composition as taught herein are chemically defined substances, not originating from animal or human tissues or fluids (e.g., serum, albumin or any other whey protein). Therefore, its characteristics are intrinsically stable in production batches. Furthermore,

the properties of the liquid composition, as taught herein, are remarkably stable over time, even under unfavorable temperature conditions and despite the absence of any antimicrobial agent. In fact, storage at 4 °C as well as at 37 °C (and also in intermediate and mixed temperature conditions) for at least 6 months does not change its optical/physical aspect nor its high efficiency in the cryopreservation of biological material, which It is an advantage for standardization and quality control purposes.

[0045] In addition, the liquid composition, as taught in this document, acts as an ice crystal inhibiting composition, which is more effective than compositions commonly used for vitrification, even under conditions with cooling and heating rates that are slower than generally recommended, as well as when using cryovials. For example, vitrification in cryovials typically has an average cooling or heating rate of about 100 °C/min (e.g., as described in Tao Wang et al., 2015, Determination of convective heat transfer coefficient at the outer surface of a cryovial being plunged into liquid nitrogen.

cryoletters. 36: 285 to 288), which is considerably less than the commonly recommended cooling or heating rate of 2000 °C to 20,000 °C per minute for vitrification purposes. When testing the vitrification ability of the liquid composition as taught in this document under conditions of high thermal inertia (e.g. solution filled French straws) and in the absence of cells, no ice crystals are optically detectable upon subsequent cooling or heating of the liquid. liquid composition for cryopreservation, while on the other hand, a reference vitrification solution and the liquid composition as taught herein devoid of PVA do not allow for consistent vitrification and result in rapid crystal formation upon cooling or heating, which can be deleterious. for biological material.

[0046] In addition to efficiency, its chemically defined composition guarantees the reproducibility of its manufacturing properties and high medium to long-term stability, which is an asset for standardization and quality control purposes. The absence of complex proteins or undefined components of human or animal origin complies with biological safety requirements.

[0047] Accordingly, a first aspect provides a liquid composition for cryopreservation of a biological material comprising, consisting essentially of, or consisting of a polar aprotic solvent, a monohydric or polyhydric alcohol, an unbranched polysaccharide, a branched polysaccharide and polyvinyl alcohol.

[0048] The term "polar aprotic solvent", as used herein, refers to a solvent consisting of polar molecules that do not contain positively charged labile hydrogen ions. Polar aprotic solvents are typically capable of forming hydrogen bonds with water to dissolve in water, while non-polar solvents are not capable of forming strong hydrogen bonds. Non-limiting examples of polar aprotic solvents are DMSO, dimethylformamide, dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, acetone and hexamethylphosphoric triamide.

[0049] In particular embodiments, the polar aprotic solvent is selected from the group consisting of dimethylsulfoxide (DMSO), dimethylformamide (DMF), preferably DMSO.

[0050] The term "monohydric alcohol", as used herein, refers to an organic compound having an alcohol functional group (i.e., a hydroxyl group (-OH)). Non-limiting examples of monohydric alcohols include methanol, ethanol, isopropyl alcohol (isopropanol), butanol and pentanol.

[0051] The term "polyhydric alcohol" as used herein refers to an organic compound having more than one alcohol functional group (i.e., more than one hydroxyl group (-OH)). Non-limiting examples of polyhydric alcohols include ethylene glycol, propylene glycol, glycerol, erythritol, sorbitol, mannitol, xylitol and volemitol.

[0052] As used herein, the singular form "monohydric or polyhydric alcohol" includes referents in both the singular and plural, unless the context clearly dictates otherwise. When monohydric or polyhydric alcohol is used to refer to a combination of different types of monohydric and/or polyhydric alcohols, the total amount of the different types of monohydric and/or polyhydric alcohols, respectively, will not be greater than the amount of the alcohol. monohydric or polyhydric in the liquid composition as taught herein. In particular embodiments, the monohydric or polyhydric alcohol is selected from the group consisting of ethylene glycol, methanol, ethanol, isopropyl alcohol (isopropanol), butanol, pentanol, propylene glycol, glycerol, erythritol, sorbitol, mannitol, xylitol and volemitol, of a preferably the monohydric or polyhydric alcohol is ethylene glycol, propylene glycol or glycerol, more preferably ethylene glycol.

[0053] The term "polysaccharide", as used herein, refers to a branched or unbranched homo- or heterodimer (i.e. 2 monosaccharides, also known as a disaccharide), oligomer or polymer (i.e. more than 20 monosaccharides, also known as polysaccharides) of monosaccharides. Monosaccharides can be combined via glycosidic bonds.

Non-limiting examples of polysaccharides as defined herein include sucrose, trehalose, lactulose, melibiose, lactobionate, raffinose, dextran, pectin, Ficoll, starch, cellulose and derivatives thereof, such as hydroxyethyl starch and methyl cellulose.

[0054] In particular embodiments, the unbranched polysaccharide is selected from the group consisting of sucrose, trehalose, lactulose, melibiose, lactobionate, raffinose and cellulose or a derivative thereof, such as methylcellulose, preferably sucrose.

[0055] In particular embodiments, the branched polysaccharide is selected from the group consisting of dextran, pectin, Ficoll and starch or a derivative thereof, such as hydroxyethyl starch, preferably dextran. In particular embodiments, the unbranched polysaccharide is sucrose and the branched polysaccharide is dextran.

[0056] The term "polyvinyl alcohol", "PVA", "PVOH" or "PVAI", as used herein, refers to the water-soluble synthetic polymer defined by the chemical formula [CH2CH(OH)]n. Polyvinyl alcohol can also be identified by CAS number 9002-89-5.

[0057] The term "cryopreservation", as used herein, refers to a process where a biological material (e.g. cells, tissues, organs, organisms) is preserved in an environment with a temperature below 0 °C , preferably below -80°C (eg using solid carbon dioxide), even more preferably below -130°C (eg using liquid nitrogen). Biological material is normally susceptible to damage caused by unregulated biochemical kinetics. At temperatures below -130 °C, any enzymatic and biochemical activity that could damage the biological material is effectively stopped. During cryopreservation procedures, the biological material to be preserved by cooling or freezing is normally placed in contact with one or more cryoprotectants. Cryopreservation can be achieved by any cryopreservation procedure known in the art, including freezing procedures such as slow freezing (SLF) and vitrification procedures such as multi-step vitrification and one-step vitrification, as described elsewhere in this document.

[0058] The term "biological material", as used herein, refers to cells, cell aggregates, tissue samples, organs, biological fluids, (multi)cellular organisms and any other membranous (e.g. liposome) , nucleotide (DNA or RNA), nucleoprotein (virus), lipid or protein entities. Non-limiting examples of biological material include stem cells (e.g., mesenchymal stem cells or induced pluripotent stem cells), gametes (e.g., sperm, oocytes, eggs), embryo (i.e., at various stages of development, for e.g. zygote, two cells, morula, blastocyst), whole blood or fractions thereof (e.g. white blood cells, red blood cells, plasma, platelets, proteins, lipids, antibodies), bone marrow, bacteria, yeasts, nematodes, membranous bodies , nucleic acids (DNA and RNA) and viruses.

[0059] In particular modalities, the biological material is selected from the group of stem cells, gametes and embryos. Preferably, the biological material is an embryo, preferably a mouse or human embryo.

[0060] The term "stem cell" generally refers to an unspecialized or relatively less specialized and proliferation-competent cell that is capable of self-renewal, that is, can proliferate without differentiation and whose progeny can give rise to to at least one relatively more specialized cell type. The term encompasses stem cells capable of substantially unlimited self-renewal, i.e., in which the progeny of a stem cell or at least part of it substantially retains the unspecialized or relatively less specialized phenotype, differentiation potential, and proliferation capacity of the stem cell. stem cell, as well as stem cells that exhibit limited self-renewal, i.e., in which the ability of the progeny or part of it for further proliferation and/or differentiation is demonstrably reduced compared to the mother cell. By way of example and not limitation, a stem cell can give rise to offspring that can differentiate along one or more lineages to produce increasingly relatively more specialized cells, in which such offspring and/or increasingly more relatively more specialized cells may be stem cells as defined herein, or even to produce terminally differentiated cells, i.e., fully specialized cells, which may be post-mitotic.

[0061] The term "induced pluripotent stem cells" or "iPS cells", as used herein, refers to pluripotent stem cells generated from adult cells by reprogramming. iPS cells can self-renew and give rise to cell types originating from all three germ layers of an organism, i.e., mesoderm, endoderm, and ectoderm, and potentially to any and all cell types in an organism, although it is not capable of to grow throughout the body. Examples of iPS cells are those taught, inter alia, by Yamanaka et al. 2006 (Cell 126: 663 to 676) and Yamanaka et al. 2007 (Cell 131: 861 to 872).

[0062] As used in this document, the qualifier "pluripotent" denotes the ability of a cell to give rise to cell types originating from all three germ layers of an organism, i.e. mesoderm, endoderm and ectoderm, and potentially capable of give rise to any and all types of cells in an organism.

[0063] In particular embodiments, the stem cells are multipotent stem cells selected from the group consisting of mesenchymal stem cells (MSCs), blood-derived stem cells (BDSCs), stem cells derived from umbilical cord blood ( UCBSCs) and bone marrow-derived stem cells (BMSCs). In preferred embodiments, the cells are MSCs.

[0064] The term "mesenchymal stem cell" or "MSC", as used herein, refers to an adult stem cell derived from mesoderm that is capable of generating cells from mesenchymal lineages, typically from two or more lineages. mesenchymal lineages, more typically three or more mesenchymal lineages, e.g. chondro-

osteoblastic (bone and cartilage), osteoblastic (bone), chondroblastic (cartilage), myocytic (muscle), tenocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) and stromogenic (medullary stroma).

MSC can be isolated from a biological sample, preferably a biological sample from a human subject, for example bone marrow, trabecular bone, blood, umbilical cord, placenta, fetal yolk sac, skin (dermis), specifically fetal skin and adolescent, periosteum, dental pulp, tendon and adipose tissue.

[0065] The term "MSC" also encompasses progeny of MSCs, eg progeny obtained by in vitro or ex vivo proliferation (propagation/expansion) of MSCs obtained from a biological sample of an animal or human subject.

[0066] In particular embodiments, the biological material is a biological sample. The term "biological sample" or "sample" as used herein refers to a sample obtained from a biological source, for example from an organism, such as an animal or human subject, cell culture, sample of fabric, etc. A biological sample from an animal or human subject refers to a sample taken from an animal or human subject and comprising cells therefrom. The biological sample from an animal or human subject may comprise one or more tissue types and may comprise cells from one or more tissue types. Methods of obtaining biological samples from an animal or human subject are well known in the art, for example tissue biopsy or blood collection. Human MSC, its isolation, in vitro expansion and differentiation have been described in, for example, Patent No. US 5,486,359; Patent No.

US 5,811,094; Patent No. US 5,736,396; Patent No.

US 5,837,539; or Patent No. US 5,827,740.

[0067] The term "subject", "donor" or "patient", as used herein, refers to animals, preferably warm-blooded animals, more preferably vertebrates, even more mammals, even more preferably primates, and specifically includes humans and non-human mammals and primates. Preferred individuals are humans.

[0068] In particular embodiments, the biological material is one or more mammalian cells, preferably mouse or human cells, even more preferably human cells.

[0069] In a particular embodiment, the biological material is biological material for assisted reproductive technologies (ART), such as oocytes and metaphase II (MII) embryos (eg, embryos at various stages of development).

[0070] In particular embodiments, the composition further comprises a diluent, preferably at pH 7.2 to 7.4.

[0071] In particular embodiments, said diluent is balanced saline, preferably phosphate buffered saline (PBS), more preferably Dulbecco's PBS (D-PBS).

[0072] The term "a balanced salt solution" or "BSS", as used herein, refers to a solution made at a physiological pH (preferably around pH 7.4) and isotonic salt concentrations. . BSS normally comprises sodium, potassium, calcium, magnesium and chloride. Non-limiting examples of balanced saline solutions include PBS, HEPES, Alsever's solution, Earle's balanced saline solution (EBSS), Gray's balanced saline solution (GBSS), Hank's balanced saline solution (HBSS), Ringer's balanced saline solution (RBSS) ) and Tyrode's balanced salt solution (TBSS).

[0073] In particular embodiments, said diluent does not comprise any proteins, polypeptides or peptides.

[0074] Liquid compositions as taught herein are effective for cryopreservation of biological material. First, the combination of specific reagents, more particularly monohydric or polyhydric alcohol, unbranched polysaccharide, branched polysaccharide and polyvinyl alcohol, which provides optimal cryopreservation.

Furthermore, the inventors have identified an optimal ratio of these different components of the polar aprotic solvent that is effective over a wide range of concentrations.

Consequently, a stock solution (i.e. undiluted 1x concentrated solution) can be prepared which can be further diluted into compositions with lower concentrations of the polar aprotic solvent, monohydric or polyhydric alcohol, unbranched polysaccharide, branched polysaccharide and polyvinyl alcohol. For use.

[0075] Accordingly, the invention provides liquid compositions comprising a polar aprotic solvent, a monohydric or polyhydric alcohol, an unbranched polysaccharide, a branched polysaccharide and polyvinyl alcohol and their use in the cryopreservation of a biological material.

[0076] In particular embodiments, the composition comprises 0.30 to 4.0% (w/v), 0.50 to 3.50% (w/v), 0.50 to 3.0% (w/v ), 0.50 to 2.50% (w/v), 0.50 to 2.0% (w/v), 0.50 to 1.50% (w/v) or 0.50 to 1, 0% (w/v), preferably 0.30 to 4.0% (w/v), of polyvinyl alcohol. In particular embodiments, the amount of the unbranched polysaccharide in the composition is each at least 20 times that of the polyvinyl alcohol. In particular embodiments, the amount of the unbranched oligosaccharide in the composition is each at least 8 times that of the branched oligosaccharide.

[0077] In particular embodiments, the liquid composition or liquid stock composition (i.e. 1x concentrate, undiluted), as taught herein comprises, essentially consists of or consists of: - 13.0 to 23.0% (v /v), 15.0 to 20.0% (v/v), 16.0 to 19.0% (v/v), 17.0 to 18.0% (v/v), preferably 13.0 to 23.0% (v/v) of the polar aprotic solvent, for example about 17.7% (v/v) of the polar aprotic solvent; - 13.0 to 23.0% (v/v), 15.0 to 20.0% (v/v), 16.0 to 19.0% (v/v), 17.0 to 18.0 % (v/v), preferably 13.0 to 23.0% (v/v), of the monohydric or polyhydric alcohol, for example about 17.7% (v/v) of the monohydric or polyhydric alcohol ; - 9.0 to 31.0% (w/v), 10 to 30% (w/v), 15 to 25% (w/v) or 20 to 25% (w/v) of the unbranched polysaccharide, for for example, about 22.7% (w/v) of the unbranched polysaccharide; - 0.60 to 22.0% (w/v), 1.0 to 20.0% (w/v), 1.0 to 15.0% (w/v), 1.0 to 10.0 % (w/v), 1.0 to 5.0% (w/v), 2.0 to 5.0% (w/v) or 2.0 to 3.0% (w/v) of the polysaccharide branched, for example about 2.64% (w/v) of the branched polysaccharide; - 0.30 to 4.0% (w/v), 0.50 to 3.50% (w/v), 0.50 to 3.0% (w/v), 0.50 to 2.50 % (w/v), 0.50 to 2.0% (w/v), 0.50 to 1.50%

(w/v) or 0.50 to 1.0% (w/v), preferably 0.30 to 4.0% (w/v), of polyvinyl alcohol, for example about 0.88 % (w/v) of polyvinyl alcohol; and - up to 100% (v/v) of a diluent, preferably a balanced salt solution.

[0078] In particular embodiments, the liquid composition or liquid stock composition (i.e. 1x concentrated, undiluted) comprises, essentially consists of or consists of: - at least 15% (v/v), at least 16% ( v/v) or at least 17% (v/v) of the polar aprotic solvent, - at least 15% (v/v), at least 16% (v/v) or at least 17% (v/v) of the monohydric or polyhydric alcohol, - at least 15% (w/v), at least 16% (w/v), at least 17% (w/v), at least 18% (w/v), at least 19% (w/v), at least 20% (w/v), at least 21% (w/v) or at least 22% (w/v), of the unbranched polysaccharide; - at least 2.0% (w/v), at least 2.10% (w/v), at least 2.20% (w/v), at least 2.30% (w/v), at least at least 2.40% (w/v), at least 2.50% (w/v) or at least 2.60% (w/v) of the branched polysaccharide, and - about 0.9% (w/v) ) of polyvinyl alcohol.

[0079] In particular embodiments, the composition may be a dilution of a stock composition in the same diluent as the stock composition as described above. This dilution can be 1:2 (i.e. 50%), 1:3 (i.e. about 33.3%), 1:4 (i.e. 25%), 1:5 (i.e. 20% ), 1:6 (that is, about 16.7%), 1:7 (that is, about 14.3%), 1:8 (that is, about 12.5%), 1:9 (that is, about 14.3%), 1:8 (that is, about 12.5%), i.e. around 11.1%), 1:10 (ie around 10%), 1:11 (ie around 9.1%), 1:12 (ie around 8.3%) , 1:13 (that is, about 7.7%), 1:14 (that is, about 7.1%), 1:15 (that is, about 6.7%) or 1:16 ( i.e. about 6.3%) dilution in a diluent as described above, preferably a balanced salt solution.

[0080] In particular embodiments, the liquid composition (e.g. a dilution of a stock composition as described herein) comprises, essentially consists of or consists of: - at least 0.8% (v/v), at least 1% (v/v), at least 1.2% (v/v), at least 1.6% (v/v), at least 2% (v/v), at least 4.0% (v/v) /v) or at least 6.0% (v/v) of the polar aprotic solvent; - at least 0.8% (v/v), at least 1% (v/v), at least 1.2% (v/v), at least 1.6% (v/v), at least 2 % (v/v), at least 4.0% (v/v) or at least 6.0% (v/v) of the monohydric or polyhydric alcohol; - at least 0.5% (w/v), at least 1% (w/v), at least 2% (w/v), at least 4% (w/v), at least 6% (w/v) v), at least 8% (w/v), at least 10% (w/v), at least 12%

(w/v), at least 14% (w/v), at least 16% (w/v), at least 18% (w/v), at least 20% (w/v), at least 21% (w/v) or at least 22% (w/v) of the unbranched polysaccharide; - at least 0.035% (w/v), at least 0.0750% (w/v), at least 0.150% (w/v), at least 0.30% (w/v), at least 0.60 % (w/v), at least 1.0% (w/v), at least 1.50% (w/v), at least 2.0% (w/v), at least 2.10% ( w/v), at least 2.20% (w/v), at least 2.30% (w/v), at least 2.40% (w/v), at least 2.50% (w/v) v) or at least 2.60% (w/v) of the branched polysaccharide; - at least 0.015% (w/v), at least 0.1% (w/v), at least 0.2% (w/v), at least 0.3% (w/v), at least 0 .4% (w/v), at least 0.5% (w/v), at least 0.6% (w/v), at least 0.7% (w/v) or at least 0.8 % (w/v) of the polyvinyl alcohol; and - up to 100% (v/v) of a diluent, preferably a balanced salt solution.

[0081] In particular embodiments, - the polar aprotic solvent is selected from the group consisting of DMSO and dimethylformamide (DMF), - the monohydric or polyhydric alcohol is selected from the group consisting of ethylene glycol, methanol, ethanol, alcohol isopropyl (isopropanol), butanol, pentanol, propylene glycol, glycerol, erythritol, sorbitol, mannitol, xylitol and volemitol, preferably ethylene glycol, propylene glycol or glycerol, more preferably ethylene glycol; - the unbranched polysaccharide is selected from the group consisting of sucrose, trehalose, lactulose, melibiose, lactobionate, raffinose and cellulose or a derivative thereof, such as methylcellulose, preferably sucrose; and/or - the branched polysaccharide is selected from the group consisting of dextran, pectin, Ficoll and starch or a derivative thereof, such as hydroxyethyl starch, preferably dextran.

[0082] Preferably, the invention provides a liquid composition for cryopreservation of a biological material comprising, consisting essentially of or consisting of DMSO, ethylene glycol, sucrose, dextran and polyvinyl alcohol.

[0083] The term "dimethylsulfoxide" or "DMSO", as used herein, refers to an organosulfur compound having the chemical formula (CH3)2SO. The DMSO can be identified by the CAS number 67-68-5.

[0084] The term "ethylene glycol", "EG" or "1,2-ethanediol", as used herein, refers to an organic compound with the chemical formula (CH 2 OH) 2 . Ethylene glycol can be identified by CAS number 107-21-

1.

[0085] The term "sucrose" or "sucrose" or "sugar" or "SUC", as used herein, refers to a disaccharide composed of glucose and fructose with the chemical formula C12H22O11. Sucrose can be identified by the CAS number 57-50-1.

[0086] The term "dextran", as used herein, refers to a complex branched glucan polysaccharide derived from the condensation of glucose.

Dextran can be identified by the CAS number 9004-54-0.

Dextran chains can be of varying lengths (e.g. from 1 to 2000 kDa). Different types of dextran can be indicated by their molecular weight (MW). For example, non-limiting examples of variable MW dextrans include dextran 1 (i.e. with a MW of about 1000 Da), dextran 5 (i.e. with a MW of about 5000 Da), dextran 12 (i.e. with a MW of approx.

12,000 Da), dextran 25 (i.e. with a MW of about

25,000 Da), dextran 40 (i.e. with a MW of about

40,000 Da), dextran 50 (i.e. with a MW of about

50,000 Da), dextran 60 (i.e. with a MW of about

60,000 Da), dextran 70 (i.e. with a MW of about

70,000 Da), dextran 80 (i.e. with a MW of about

80,000 Da), dextran 150 (i.e. with a MW of about

150,000 Da) and dextran 200 (that is, with a MW of about

200,000 Da).

[0087] As used in this document, the singular form "dextran" includes both singular and plural referents unless the context clearly dictates otherwise. When dextran is used to refer to a combination of different types of dextran, the total amount of the different types of dextran will not be greater than the amount of the branched polysaccharide in the liquid composition as taught herein.

[0088] In particular embodiments, the dextran present in the liquid composition as taught herein may comprise, consist essentially of or consist of only one type of dextran or a combination of different types of dextran, such as a combination of dextrans with a different MW .

[0089] In particular embodiments, the dextran present in the liquid composition as taught herein may comprise, consist essentially of, or consist of only one type of dextran. In more particular embodiments, the dextran present in the liquid composition as taught herein comprises, consists essentially of or consists of dextran 1, dextran 40 or dextran 70. For example, the liquid composition as taught herein comprises, consists essentially of or consists of dextran 1.

[0090] In preferred embodiments, the dextran present in the liquid composition, as taught herein, comprises a combination of dextrans of different molecular weights.

[0091] In more preferred embodiments, the dextran essentially consists of or consists of dextran 1, dextran 40 and dextran 70.

[0092] The ratio between the amounts of different types of dextran (eg, dextrans of different molecular weight) in the composition is not critical for the liquid composition of the invention.

[0093] However, in particular embodiments, the dextran essentially consists of or consists of dextran 1, dextran 40 and dextran 70 in a ratio of 1:1:1, 2:1:1, 10:10:1 or 10: 1:10, preferably a ratio of 1:1:1. An example of dextran 1 is dextran 1 from Pharmacosmos (ref. 5510 0001 1007). An example of dextran 40 is dextran 40 from AppliChem (ref. A2249). An example of dextran 70 is Dextran 70 from AppliChem (ref.

A1847).

[0094] In particular embodiments, the liquid composition comprises substantially equal amounts of DMSO and ethylene glycol and a sucrose concentration that is at least 8 times that of dextran.

[0095] In particular modalities, the liquid composition comprises:

- substantially equal amounts of DMSO and ethylene glycol, - a sucrose concentration that is at least 8 times that of dextran, and - a sucrose concentration that is at least 20 times that of polyvinyl alcohol.

[0096] In particular embodiments, the liquid composition (for example, a dilution of a stock composition as described in this document) comprises: - at least 0.8% (v/v), at least 1% (v/v) , at least 1.2% (v/v), at least 1.6% (v/v), at least 2% (v/v), at least 4.0% (v/v) or at least 6 .0% (v/v) DMSO; - at least 0.8% (v/v), at least 1% (v/v), at least 1.2% (v/v), at least 1.6% (v/v), at least 2 % (v/v), at least 4.0% (v/v) or at least 6.0% (v/v) ethylene glycol; - at least 0.5% (w/v), at least 1% (w/v), at least 2% (w/v), at least 4% (w/v), at least 6% (w/v) v), at least 8% (w/v), at least 10% (w/v), at least 12% (w/v), at least 14% (w/v), at least 16% (w/v) v), at least 18% (w/v), at least 20% (w/v), at least 21% (w/v), at least 22% (w/v) sucrose; - at least 0.037% (w/v), at least 0.075% (w/v), at least 0.15% (w/v), at least 0.2% (w/v), at least

0.25% (w/v), at least 0.5% (w/v), at least 1% (w/v), at least 1.25% (w/v), at least 1.5% (w/v), at least 1.75% (w/v), at least 2% (w/v), at least 2.25% (w/v) or at least 2.5% (w/v) ) of dextran; - at least 0.015% (w/v), at least 0.1% (w/v), at least 0.2% (w/v), at least 0.3% (w/v), at least 0 .4% (w/v), at least 0.5% (w/v), at least 0.6% (w/v), at least 0.7% (w/v) or at least 0.8 % (w/v) of polyvinyl alcohol; and - up to 100% (v/v) of a diluent, preferably with pH 7.2 to 7.4.

[0097] In particular modalities, the liquid composition or liquid stock composition (i.e. 1x concentrated, undiluted) comprises, essentially consists of or consists of: - 13.0 to 23.0% (v/v), 15 .0 to 20.0% (v/v), 16.0 to 19.0% (v/v), 17.0 to 18.0% (v/v) or 13.0 to 23.0% ( v/v) DMSO, for example about 17.7% (v/v) DMSO; - 13.0 to 23.0% (v/v), 15.0 to 20.0% (v/v), 16.0 to 19.0% (v/v), 17.0 to 18.0 % (v/v) or 13.0 to 23.0% (v/v) ethylene glycol, for example about 17.7% (v/v) ethylene glycol; - 9.0 to 31.0% (w/v), 10.0 to 30.0% (w/v), 15.0 to 30.0% (w/v), 15.0 to 25.0 % (w/v), 20.0 to 25.0% (w/v) or 22.0 to 23.0% (w/v), for example about 22.7% (w/v) of sucrose;

- 0.60 to 22.0% (w/v), 1.0 to 20.0% (w/v), 1.0 to 15.0% (w/v), 1.0 to 10.0 % (w/v), 1.0 to 7.50% (w/v), 1.0 to 5.0% (w/v), 2.0 to 4.0% (w/v) or 2 .0 to 3.0% (w/v), for example about 2.64% (w/v) dextran; - 0.30 to 4.0% (w/v), 0.50 to 3.5% (w/v), 0.50 to 3.0% (w/v), 0.50 to 2.50 % (w/v), 0.50 to 2.0% (w/v), 0.50 to 1.50% (w/v) or 0.50 to 1.0% (w/v) of alcohol polyvinyl, for example about 0.88% (w/v) polyvinyl alcohol; and/or - up to 100% (v/v) of a diluent, preferably with pH 7.2 to 7.4.

[0098] In particular embodiments, the composition or stock composition comprises, essentially consists of or consists of at least 15% (v/v) DMSO, at least 15% (v/v) ethylene glycol, at least 15% ( w/v) sucrose, at least 2.5% dextran (w/v), about 0.9% (w/v) polyvinyl alcohol and a diluent up to 100% (v/v).

[0099] In particular embodiments, the composition or stock composition comprises, essentially consists of or consists of about 17.7% (v/v) DMSO, about 17.7% (v/v) ethylene glycol, about 22.7% (w/v) sucrose, about 0.88% dextran 1 (w/v), about 0.88% dextran 40 (w/v), about 0.88% dextran 70 (w/v) /v), about 0.88% (w/v) polyvinyl alcohol and D-PBS up to 100% (v/v).

[00100] In particular embodiments, the liquid composition does not comprise any proteins, polypeptides or peptides, preferably the liquid composition does not comprise any proteins, polypeptides or peptides of mammalian origin. For example, the liquid composition does not contain albumin or other serum proteins.

[00101] In particular embodiments, the liquid composition does not comprise animal components.

[00102] In particular embodiments, the liquid composition does not comprise serum.

[00103] In particular embodiments, the liquid composition does not comprise antimicrobial agents such as antibiotics.

[00104] The present inventors have found that the liquid composition as taught herein allows for highly efficient cryopreservation (e.g., vitrification or freezing) of biological material with similar viability, proliferation, and morphological endpoints to non-cryopreserved biological control material.

[00105] Accordingly, another aspect provides the use of the liquid composition, as taught herein, for the cryopreservation of a biological material.

[00106] In particular embodiments, cryopreservation of biological material is performed by a freezing procedure, such as a slow freezing procedure, or a vitrification procedure, such as multi-step vitrification or one-step vitrification.

[00107] The term "freezing or slow freezing procedure" or "SLF", as used herein, refers to a process of cooling biological material to a temperature below 0 °C, for example, at a temperature of at least -80 °C or at least -196 °C, where the biological material is cooled at a slow, controlled rate - for example, at a decrease of 0.1 °C to 2 °C per minute. The controlled rate of cooling can be achieved by any device known in the art for such purposes, such as a controlled rate freezer or a portable bench top freezing vessel. The biological material is normally placed in contact with one or more cryoprotectants (e.g. 10% (v/v) DMSO in culture medium) to prevent - e.g. mechanical rupture of membranes due to ice crystals - and osmotic damage. in the cell.

[00108] For example, a slow freezing procedure may comprise the following steps: immersion of a biological material in a cryopreservation solution containing 10% dimethyl sulfoxide (DMSO) (e.g. CryoStor® CS10 (STEMCELL Technologies)) or in a diluting 50% or 25% of the liquid composition as taught herein, transferring the biological material to cryovials, storing the cryovials comprising the biological material overnight in a freezing vessel (e.g. Mr Frosty™ device (Nalgene)) in a -80 °C freezer and subsequently transferring the cryovials into liquid nitrogen.

[00109] The term "vitrification", as used herein, refers to a process of cooling biological material, in which the biological material is cooled to a temperature below 0 °C, for example, to a temperature of at least -130 °C, at a very rapid cooling rate - for example, from 2000 °C to

20,000 °C per minute - until reaching the final storage temperature. The biological material is typically placed in contact with one or more cryoprotectants (typically 5 to 7.15 M) to prevent osmotic damage to the cell and inhibit ice formation in the cooling process. Vitrification procedures are known in the art and include multi-step and one-step vitrification procedures. Vitrification procedures normally comprise at least one step of dehydrating the biological material with an aqueous solution ("vitrification solution") containing permeating and/or non-permeating cryoprotectants. The biological material together with an amount of vitrification solution is placed in a suitable cryocontainer and cooled rapidly by immersion in a cryogenic fluid, such as liquid nitrogen or vapor thereof. A well-balanced cooling rate and concentration of cryoprotectants allow intracellular water to reach a solid, innocuous, glassy state rather than an orderly and harmful crystalline ice state. Vitrification devices are known in the art and include, for example, cryocontainers.

[00110] For example, a one-step vitrification procedure may comprise the following steps: contacting a biological material with the undiluted liquid composition for cryopreservation as taught herein and moving it in the composition to eliminate substantially all of the culture medium in around the biological material, harvesting the biological material into a smaller volume of the liquid composition as taught in this document and loading it into a protective straw or cryotube. The straw or cryovial is sealed and dipped directly into liquid nitrogen. The period between the transfer of biological material to the liquid composition, as taught in this document, and the immersion in liquid nitrogen is a maximum of 1 minute.

[00111] For example, a multi-step (equilibration) vitrification procedure may comprise the following steps: subsequently immersing the biological material in decreasing dilutions of the liquid cryopreservation stock composition as taught in this document (e.g. 3 minutes in a dilution of 1:16,

3 minutes at a 1:8 dilution, 3 minutes at a 1:4 dilution, 5 minutes at a 1:2 dilution), immersing the biological material in a smaller volume of the undiluted stock composition as taught in this document at a protective straw or cryotube. The straw or cryovial is sealed and dipped directly into liquid nitrogen.

[00112] In particular embodiments, the liquid composition as taught herein is used for cryopreservation of a biological material at a temperature of at least -80°C, at least -70°C, at least -80°C, at least - 90 °C, at least -100 °C, at least -110 °C, at least -120 °C, at least -130 °C, at least -140 °C, at least -150 °C, at least - 160 °C, at least -170 °C, at least -180 °C, at least -190 °C or at least -200 °C.

[00113] In particular embodiments, the liquid composition as taught herein is used for cryopreservation of a biological material for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years.

[00114] Generally recommended cooling and heating rates for cryopreservation biological materials are [~20,000 °C/min]. In particular embodiments, the cooling and/or heating rate of the vitrification method is [~1000 °C/min]. The inventors have found that the compositions of the present invention provide strong ice crystal inhibiting properties in vitrification methods where cooling rates and/or heating rates are [~1000°C/min].

[00115] Likewise, another aspect provides a method for preserving a biological material comprising the steps of: - providing a biological material; - contacting biological material with liquid composition (eg stock composition or stock composition dilution) as taught in this document; - cooling and/or heating the biological material in the liquid composition, as taught herein.

[00116] The term "contact" or "contact" as used herein refers to bringing one or more first components (such as one or more molecules, biological entities, cells or materials) together with one or more second components ( as one or more molecules, biological entities, cells or materials) in such a way that the first component(s) is/are directly exposed (i.e. in direct contact with) the second(s) ) component(s) and may - if able to do so - link or modulate the second component(s) or that the second component(s) ) can - if capable - link or modulate the first component(s). The term "contact" can, depending on the context, be synonymous with "expose", "incubate", "mix" or the like.

[00117] In particular embodiments, contacting the biological material with the liquid composition, as taught herein, is achieved by immersing or dipping the biological material in a volume of the liquid composition, preferably wherein the volume of the liquid composition is sufficient to completely envelop the biological material. The skilled person will understand that the volume of liquid composition used depends on the type and amount of biological material. For example, if the biological material is an embryo, 0.1 µl to 1 µl of the liquid composition may be sufficient.

[00118] In particular embodiments, contact of the biological material with the liquid composition is carried out in a container that is suitable for cryopreservation. Such containers are known in the art and include, for example, a protective straw or a cryovial.

[00119] In particular embodiments, the method comprises one or more pre-cooling equilibration steps, which involve brief (30 to 600 s, preferably 180 to 300 s) contact with one or more (usually increasing) concentrations of the vitrification medium at a temperature between 15 and 37 °C. Pre-cooling equilibration steps are typically included in multi-step cooling procedures, such as multi-step vitrification, as described elsewhere in this document.

[00120] In particular embodiments, the methods involve cooling the biological material. In other embodiments, the methods involve heating the biological material. In particular embodiments, the biological material is cooled to a temperature below the freezing point and, after a certain period of time, heated to a temperature above the freezing point.

[00121] In contrast to commonly used methods to preserve a biological material, including a cooling step (e.g. vitrification), making use of several different compositions (i.e. consisting of different ingredients) to balance the biological material prior to cooling to achieve satisfactory process efficiency, the liquid composition as taught in this document allows the use of the same stock solution (but at various dilutions) to balance the biological material.

[00122] Therefore, in particular embodiments, the method comprises contacting the biological material with at least one (such as one, two, three or four, preferably four) dilution of the liquid composition (e.g. stock composition) as taught herein before contacting the biological material with the undiluted liquid composition (e.g. stock composition) as taught herein, wherein said step is performed prior to cooling the biological material. In more particular embodiments, the undiluted liquid stock composition comprises, consists essentially of or consists of 13.0 to 23.0% (v/v) DMSO; 13.0 to 23.0% (v/v) ethylene glycol, 9.0 to 31.0% (w/v) sucrose; 0.60 to 22.0% (w/v) dextran; 0.30 to 4.0% (w/v) of polyvinyl alcohol and up to 100% (v/v) of a diluent, preferably about 17.7% (v/v) of DMSO, about 17 .7% (v/v) ethylene glycol, about 22.7% (w/v) sucrose, about 0.88% dextran 1 (w/v), about 0.88% dextran 40 ( w/v), about 0.88% dextran 70 (w/v), about 0.88% (w/v) polyvinyl alcohol and D-PBS up to 100% (v/v).

[00123] In particular embodiments, the at least one dilution of the liquid composition (e.g., stock composition) as taught herein is a dilution of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15 and/or 1:16.

[00124] In particular embodiments, the method subsequently comprises contacting the biological material with two or more (such as two, three or four, preferably four) decreasing dilutions of the liquid composition (e.g., stock composition) as taught herein document prior to contacting the biological material with the undiluted liquid composition (e.g. stock composition) as taught herein, wherein said step is performed prior to cooling the biological material, preferably wherein the last dilution of the liquid composition with which the biological material is contacted before contacting the biological material with the undiluted liquid composition is a 1:2 dilution.

[00125] In particular embodiments, the method comprises contacting the biological material with a 1:16 dilution, a 1:8 dilution, a 1:4 dilution and a 1:2 dilution of the liquid composition (e.g., stock composition) as taught herein before contacting the biological material with said composition (e.g. stock composition), wherein said step is performed prior to cooling the biological material, preferably wherein the undiluted liquid stock composition comprises, consists essentially of or consists of 13.0 to 23.0% (v/v) DMSO; 13.0 to 23.0% (v/v) ethylene glycol, 9.0 to 31.0% (w/v) sucrose; 0.60 to 22.0% (w/v) dextran; 0.30 to 4.0% (w/v) of polyvinyl alcohol and up to 100% (v/v) of a diluent, more preferably about 17.7% (v/v) of DMSO, about 17.7% (v/v) ethylene glycol, about 22.7%

(w/v) sucrose, about 0.88% dextran 1 (w/v), about 0.88% dextran 40 (w/v), about 0.88% dextran 70 (w/v) v), about 0.88% (w/v) polyvinyl alcohol and D-PBS up to 100% (v/v).

[00126] In particular embodiments, the method comprises contacting the biological material with - a 1:16 dilution of the liquid composition (e.g., stock composition) as taught herein for at least 0.5 minute, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes or at least 3 minutes, preferably at least 3 minutes, - a 1:8 dilution of the liquid composition (e.g. stock composition) as taught in this document for at least 0.5 minute, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes or at least 3 minutes, preferably at least 3 minutes, - a 1:4 dilution of the liquid composition (e.g., stock composition) as taught herein for at least 0.5 minute, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, or at least 3 minutes, of a preferably at least 3 minutes, and - a 1:2 dilution of the composition liquid (for example,

stock composition) as taught herein for at least 0.5 minute, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes or at least 3 minutes, preferably at least 5 minutes , before contacting the biological material with said composition (e.g. stock composition), wherein said step is performed prior to cooling the biological material.

[00127] In particular modalities, each step of contacting the biological material with the 1:16 dilution of the liquid composition, the 1:8 dilution of the liquid composition, the 1:4 dilution of the liquid composition and the 1:2 dilution of the liquid composition it is carried out for a period of 0.5 to 10 minutes, from 1 to 6 minutes or from 3 to 5 minutes, preferably from 3 to 5 minutes.

[00128] In particular embodiments, the cooling of the biological material is carried out within 3 minutes, 2 minutes or 1 minute, preferably within 1 minute, of contacting the biological material with the liquid composition (i.e., diluted or undiluted) as taught in this document.

[00129] In particular embodiments, said cooling comprises lowering the temperature of the biological material to a temperature of at least -70°C, at least -80°C, at least -90°C, at least -100°C, at least at least -110 °C, at least -120 °C, at least -130 °C, at least -140 °C, at least -150 °C, at least -160 °C, at least -170 °C, at least -180 °C, at least -190 °C or at least -200 °C. This can be accomplished by any methods known in the art, such as freezing procedures (e.g. slow freezing) or vitrification.

[00130] In particular embodiments, said cooling is performed by a slow freezing procedure. In more particular embodiments, if cooling is carried out by a slow freezing procedure, the liquid composition is a dilution, preferably a 1:2 or 1:4 dilution, more preferably a 1:4 dilution. ; of the net inventory composition that comprises, consists essentially of or consists of 13.0 to 23.0% (v/v) of DMSO; 13.0 to 23.0% (v/v) ethylene glycol, 9.0 to 31.0% (w/v) sucrose; 0.60 to 22.0% (w/v) dextran; 0.30 to 4.0% (w/v) of polyvinyl alcohol and up to 100% (v/v) of a diluent, preferably about 17.7% (v/v) of DMSO, about 17 .7% (v/v) ethylene glycol, about 22.7% (w/v) sucrose, about 0.88% dextran 1 (w/v), about 0.88% dextran 40 ( w/v), about 0.88% dextran 70 (w/v), about 0.88% (w/v) polyvinyl alcohol and D-PBS until

100% (v/v).

[00131] In particular embodiments, said method comprises a one-step vitrification of the biological material. In particular embodiments, if the method comprises a one-step vitrification of the biological material, the biological material is merely contacted with the undiluted liquid stock composition which comprises, consists essentially of or consists of 13.0 to 23.0% (v/v) of DMSO; 13.0 to 23.0% (v/v) ethylene glycol, 9.0 to 31.0% (w/v) sucrose; 0.60 to 22.0% (w/v) dextran; 0.30 to 4.0% (w/v) of polyvinyl alcohol and up to 100% (v/v) of a diluent, preferably about 17.7% (v/v) of DMSO, about 17 .7% (v/v) ethylene glycol, about 22.7% (w/v) sucrose, about 0.88% dextran 1 (w/v), about 0.88% dextran 40 ( w/v), about 0.88% dextran 70 (w/v), about 0.88% (w/v) polyvinyl alcohol and D-PBS up to 100% (v/v).

[00132] In particular embodiments, the step of contacting the biological material with the liquid composition, as taught in this document, is carried out at a temperature from 15 °C to 40 °C, from 15 °C to 37 °C, from 20 °C C to 37 °C or from 20 °C to 30 °C. For example at about 20°C.

[00133] In particular embodiments, the temperature of the biological material before cooling the biological material is from 15 °C to 40 °C, from 15 °C to 37 °C, from 20 °C to

37 °C or from 20 °C to 30 °C. For example at about 20°C.

[00134] In particular embodiments, said method comprises storing the biological material for a period of at least 1 minute, at least 1 hour or at least 1 day, wherein said storage is carried out after cooling the biological material, and in that said storage is carried out at a temperature of at least -70 °C, at least -80 °C, at least -90 °C, at least -100 °C, at least -110 °C, at least -120 ° C, at least -130 °C, at least -140 °C, at least -150 °C, at least -160 °C, at least -170 °C, at least -180 °C, at least -190 °C or at least -200°C, preferably at least -100°C, more preferably at least -190°C.

[00135] In particular embodiments, said method comprises heating the biological material after cooling the biological material. Heating the biological material can be accomplished by any methods known in the art to heat or increase the temperature of a cooled, frozen or vitrified biological material.

[00136] In particular embodiments, the heating comprises directly contacting the cooled, preferably vitrified, biological material with a culture medium for cultivating said biological material, wherein said culture medium has a temperature of at least 25° C, at least 26 °C, at least 27 °C, at least 28 °C, at least 29 °C, at least 30 °C, at least 31 °C, at least 32 °C, at least 33 °C, at least 34°C, at least 35°C, at least 36°C or at least 37°C, eg 37°C. For example, pre-warmed culture medium (eg at 37°C) can be added directly to cooled, preferably vitrified, biological material. Subsequently, the liquid composition for cryopreservation as taught herein may be substantially separated from the thawed biological material, for example, by centrifugation.

Subsequently, the biological material can be placed in contact with fresh culture medium and placed in culture.

[00137] The term "growth medium" or "culture medium" as used herein refers to a solid, liquid, or semi-solid designed to support the growth of cells or tissue. The growth medium typically comprises macronutrients (e.g. nitrogen, phosphorus, potassium, calcium, magnesium or sulfur), micronutrients (e.g. iron, manganese, zinc, boron, copper, molybdenum), vitamins, amino acids, one or more sugars (eg glucose), inorganic salts and/or proteins (eg transferrin). Growth media are well known in the art and can vary depending on the type of cell or tissue cultured, as well as the purpose of the culture (e.g., differentiation, expansion, or maintenance). Non-limiting examples of growth media include Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), alpha-modified Minimum Essential Medium (alpha-MEM), Essential Basic Medium (BME), Iscove's Modified Dulbecco Medium ( IMDM), BGJb Medium, F-12 Nutrient Mix (Ham), Liebovitz L-15, DMEM/F-12, Essential Modified Eagle Medium (EMEM), RPMI-1640, Medium 199, MB 752/1 from Waymouth or Williams E medium, Basal Mesenchymal Stem Cell Medium and modifications and/or combinations thereof.

[00138] The present inventors have found that the liquid composition as described herein is especially beneficial in slow rate heating (e.g. including air heating or water bath heating (e.g. at a temperature of 37°C)) of biological material, such as mouse embryos, compared to a control cryopreservation solution.

[00139] In particular embodiments, heating the biological material comprises heating the cooled, preferably vitrified, biological material by exposing the cooled, preferably vitrified, biological material to a water bath having a temperature of 15°C to 37 °C (e.g. at a temperature of about 37 °C) for a period of not more than 10 seconds, before contacting the biological material with the culture medium to cultivate said biological material, wherein said culture has a temperature of at least 25 °C, at least 26 °C, at least 27 °C, at least 28 °C, at least 29 °C, at least 30 °C, at least 31 °C, at least 32 °C, at least 33 °C, at least 34 °C, at least 35 °C, at least 36 °C or at least 37 °C, eg 37 °C.

[00140] In particular embodiments, heating the biological material comprises contacting the biological material with at least one hyperosmotic solution composed of a non-penetrating cryoprotectant, such as sucrose or trehalose, before contacting the biological material with the culture medium to cultivate said biological material with a temperature of at least 25 °C.

[00141] In particular embodiments, heating the biological material comprises contacting the biological material with at least one sucrose solution before contacting the biological material with the culture medium to cultivate said biological material at a temperature of at least 25 °C , wherein at least one sucrose solution is a 1M, 0.75M, 0.6M, 0.5M, 0.25M, 0.2M and/or 0.1M sucrose solution, of preferably in a balanced salt solution, more preferably in

PBS, even more preferably in D-PBS.

[00142] In particular embodiments, heating the biological material subsequently comprises contacting the biological material with a 1 M, 0.75 M, 0.5 M and 0.25 M sucrose solution before contacting the biological material with the culture for cultivating said biological material at a temperature of at least 25°C.

[00143] In particular embodiments, the methods taught herein allow the biological material to remain viable after being heated (eg, heated or thawed).

[00144] While the invention has been described in conjunction with its specific embodiments, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the foregoing description. Therefore, it is intended to encompass all alternatives, modifications and variations as follows in the essence and broad scope of the appended claims.

[00145] The following examples are provided to better illustrate particular embodiments and should not be considered as limiting of the order. The order is limited by the claims only.

EXAMPLES Example 1. The compositions of the present invention make it possible to successfully cryopreserve cells, gametes and embryos

Materials and methods Composition of the cryopreservation solution (CDCS)

[00146] The Chemically Defined Cryopreservation Solution (CDCS) is composed as indicated in table 1 for its reference stock concentration (1x concentrate). According to protocols, this core solution can be used undiluted or diluted to meet unique methodological/cryobiological requirements. The amounts of each of the CDCS components in the undiluted stock may vary relatively from one another within the ranges indicated in Table 1, without significantly altering the conditioning or cryoprotection properties.

Table 1: Composition of CDCS Stock Solution (1x Concentrated) Relative Ranges Component Preferred Final Composition D-PBS or Osmotically and Physiologically Solution Diluent Equivalent Diluent Dimethylsulfoxide 17.7% v/v 13.3 to 22.1% v/v (DMSO ) Ethylene glycol (EG) 17.7% v/v 13.3 to 22.1% v/v Sucrose (SUC) 22.7% w/v 9.1 to 30.3% w/v Dextran 1 (D1) 0.88% w/v 0.2 to 8.8% w/v Dextran 40 (D40) 0.88% w/v 0.2 to 8.8% w/v Dextran 70 (D70) 0.88% w/v 0.2 to 8.8% w/v Alcohol 0.88% w/v 0.3 to 3.5% w/v Polyvinyl (PVA) Composition of the reference vitrification solution (VSE)

[00147] A commonly used reference embryo vitrification solution (VSE, Vanderzwalmen et al., 2013) has been used as a point of comparison for various properties of CDCS. Table 2 compares the compositions of CDCS and VSE. This reference solution is very similar to commercial solutions commonly used in TRA (eg FertiVit™ by Fertipro).

Table 2: Comparison of CDCS Stock Solution (1x Concentrated) and VSE CDCS (1x), VSE Component, Final Composition Final Composition D-PBS or Osmotically Solution and Diluent Physiologically Equivalent Diluent Dimethylsulfoxide 17.7% v/v 20% v/v (DMSO) Ethylene glycol (EG) 17.7% v/v 20% v/v Sucrose (SUC) 22.7% w/v 17.1% w/v Dextran 1 (D1) 0.88% w/v - Dextran 40 (D40) 0.88% w/v - Dextran 70 (D70) 0.88% w/v - Alcohol 0.88% w/v - Polyvinyl (PVA) Ficoll 400 - 1% w/v Fetal serum from - 10% v/v veal (FCS) - 0.005% w/v sulfate streptomycin - 0.01% w/v monosulfate kanamycin Physical test of the vitrification capacity of CDCS

[00148] The vitrification ability of undiluted CDCS (ie, its solidification without crystal formation) after cooling and the absence or formation of crystals after subsequent heating was evaluated using a physical test. VSE, CDCS(-) (a solution closely related to CDCS, but without PVA, which was developed by us as a less efficient precursor) and CDCS were aspirated with a syringe into 250 l French semen straws (Minitube ref. 13407 /0010) that were later closed with a plug at their lower end. The straws were dipped and shaken in liquid nitrogen (LN2) and then heated in a water bath at 37 °C. The vitrification/crystallization state after cooling and subsequent heating was assessed by visual examination: a solution subjected to cooling or heating along its glass transition temperature remains clear and transparent after vitrification, while crystallization induces a white appearance. /milky.

CDCS stability tests

[00149] The CDCS was subjected to stability tests when kept (i) at 4 °C, (ii) at room temperature (22 +/- 4 °C), (iii) for two weeks at room temperature followed by storage at room temperature. 4°C, (iv) at 37°C and (v) for two weeks at 37°C followed by storage at 4°C. Aliquots were tested one day after manufacture (D1) and subsequently at various time points spanning day 7 (D7), D14 and after one (M1), two (M2), three (M3) and six (M6) months. The parameters recorded were (i) normal/abnormal appearance after visual examination (turbidity, precipitates...) and (ii) one-step murine embryo vitrification efficiency, as described below (FVB/N strain).

Cryopreservation of embryos, oocytes and cells with CDCS Mouse embryos Production and culture

[00150] Inbred C57BL6/JRj mice (Janvier Labs, France) were used in this example. Fertilized oocytes were provided by the Central Mouse Facility at the University of Liège. Zygotes with two pronuclei and normal-appearing cytoplasm were kept for later use. If applicable, they were cultured in drops of 50 l of M16 medium under mineral oil, in a saturated atmosphere of water at 37 °C under 5% CO2 until treatment. Embryos at the 2-cell stage were subjected to vitrification or maintained as non-cryopreserved controls.

glazing

[00151] Embryo vitrification was performed using a one-step method using CDCS or VSE disclosed above. Briefly, embryos are harvested from their culture medium and transferred to a 500 µl drop of CDCS or VSE. They are moved several times into the vitrification solution to remove the gangue from the culture medium. They are then collected in 0.3 to 1 l of the vitrification medium in a carrier (VitriVet™, Vitrimed, Bregenz, Austria), which is inserted into a protective straw. The straw is sealed and directly dipped and shaken in LN2.

From the transfer of embryos to CDCS or ESV to their immersion in LN2, it takes no more than 60 seconds.

Heating

[00152] Heating was performed at least 24 hours after cooling. The VitriVet™ carrier was extracted from its protective straw while still hanging from the LN2. The VitriVet™ trough is either (i) gently shaken in the open air for 10 seconds and subsequently immersed in a hot culture medium (37 °C), or (ii) directly immersed in the culture medium without the heating step of the air.

Culture and parameters

[00153] The parameters recorded were early survival and development to the blastocyst stage. Early survival was assessed by evaluating the morphology of the zona pellucida and blastomeres one hour after heating. Development to the blastocyst stage was observed and recorded on day 5 of development (D5).

Metaphase 2 mouse oocytes (MII)

[00154] FVB/NRj inbred mice and

C57BL6/JRj (Janvier Labs, France) were used in this study. MII oocytes were collected, subjected to the same vitrification and heating as the embryos, but only with CDCS and direct heating in the culture medium.

Subsequently, they underwent in vitro fertilization (IVF) with frozen sperm. Fertilized oocytes were cultured for 48 hours to 5 days. Two-cell stage embryos obtained after 48 hours were allowed to continue their development to the blastocyst stage (D5) or transferred into the oviduct of B6CBAF1/JRj pseudopregnant mothers (Janvier Labs, France) using standard procedures (Nagy A. ; Vintersten K.; Behringer R.

2003). Successful fertilization, development to 2-cell and blastocyst stages, and birth rates after transfer were recorded as parameters.

Human embryos and MII oocytes

[00155] The vitrification of embryos at the aneuploid blastocyst stage and of human MII oocytes was carried out in compliance with the legal and ethical regulations in force. CDCS was used at various dilutions in D-PBS and in undiluted form, following a multi-step equilibration protocol prior to cooling. After heating, multi-step equilibration procedures in SUC solutions were applied prior to culturing.

blastocysts

[00156] Twenty aneuploid blastocysts previously vitrified and heated using a classical procedure were subsequently revitrified with CDCS using the VitriSafe® closed carrier system (VitriMed, Austria).

Prior to cooling, the embryos were equilibrated in various dilutions (in D-PBS) of CDCS and finally in undiluted CDCS. Briefly, blastocysts were immersed for 5 minutes in 50 µl of 25% (v/v) CDCS followed by 4 to 6 minutes in 50 µl of 50% (v/v) CDCS. Subsequently, the blastocysts were transferred in a drop of undiluted 100 l CDCS for approximately 60 seconds, which includes the time required to load the embryos into the carrier in a microdroplet of 0.3 l to 1.0 l of CDCS , insert into a protective straw, seal and soak in LN2.

[00157] For heating, the VitriSafe® carriers were removed using an extractor tool. Carrier tips were immersed in 1 ml of 1 M SUC solution (in D-PBS) at room temperature. After 30 seconds, the blastocysts were transferred into 0.75M SUC for a further 30 seconds, followed by 1 minute in 0.5M SUC and 2 minutes in 0.25M SUC. The blastocysts were finally transferred to the appropriate culture medium. .

[00158] The morphological and “health” status of the treated blastocysts were recorded immediately after heating and after 18 hours in culture.

MII oocytes

[00159] Ten MII oocytes underwent a vitrification protocol similar to blastocysts, but with minor modifications in the pre-cooling and heating equilibrium steps. Prior to cooling, oocytes were sequentially immersed in 50 µl droplets of diluted CDCS (in D-PBS) as follows: 3 minutes in 6.25% (v/v) CDCS, 3 minutes in 12.5%, 3 minutes at 25% (v/v) and 5 minutes at 50%. Subsequently, they were transferred into a 100 µl drop of undiluted CDCS for approximately 60 seconds before being dipped in LN2 in the same manner described here above for blastocysts.

[00160] For heating, the carrier tip was immersed in 1 ml of 1 M SUC and the embryos were transferred in this solution for 1 minute, followed by 0.5 M SUC for 3 minutes, 0.25 M SUC for 2 minutes and 0.125 M SUC for 2 minutes. The oocytes were finally transferred to the appropriate culture medium. The morphological and “health” status of the treated oocytes were recorded immediately after heating and after 18 hours in culture.

equine mesenchymal stem cells

Isolation and culture

[00161] Mesenchymal stem cells isolated from equine cadavers (EC-MSCs, gift from Prof N. Antoine, U.

Liège) were maintained in DMEM + GlutaMAX (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% Pen/Strep (Gibco) at 37°C with 5% CO 2 . At 70 to 80% confluence, cells were passaged using 0.05% Trypsin-EDTA (Gibco).

Glazing and heating

[00162] For vitrification in a one-step format, cells were detached and aliquots of 106 cells were prepared. After centrifugation, the cell pellets were resuspended in 220 µl of CDCS (previously equilibrated at room temperature), transferred to 1.8 ml cryovials (Nunc) and immersed in LN2 during agitation, respecting a time interval of 30 s. between the first contact of cells with CDCS and immersion in LN2. Cells were stored in LN2 tank for at least 24 h. For each experiment, aliquots of untreated cells were seeded and analyzed, referred to as unvitrified controls.

[00163] The vitrified cells were subsequently heated by removing the cryotubes from the LN2, bringing and holding their bottom in a 37 °C water bath while directly adding 1 ml of pre-warmed (37 °C) culture medium and gently pipetting to up and down. The cell suspensions were then transferred to conical tubes containing 3 ml of complete culture medium, centrifuged, resuspended in fresh medium and seeded in 6-well plates.

Slow freezing (SLF) and thawing

[00164] For SLF, the cells were detached and evenly distributed into four groups. After centrifugation, cell pellets were resuspended in either culture medium (non-cryopreserved control), 10% DMSO in culture medium (Ref.) or in a 25% or 50% dilution of CDCS in D-PBS at a concentration of 106 cells/ml. 1 ml aliquots (10 6 cells) were then distributed into 1.8 ml cryotubes (Nunc) which were placed overnight in Mr Frosty™ devices (Nalgene) in a -80°C freezer prior to storage in LN2. Cells were stored in the LN2 tank for at least 4 h. Untreated cells were seeded and analyzed, referred to as non-cryopreserved controls.

[00165] The frozen cells were later heated by removing the cryotubes from the LN2, bringing and keeping their lower part in a water bath at 37 °C.

Immediately after thawing, 1 ml of culture medium was added and mixed by pipetting up and down. The cell suspensions were then transferred to conical tubes containing 3 ml of culture medium. Each sample was adjusted to 10 ml by the addition of culture medium, centrifuged, resuspended in 1 ml of fresh medium and seeded in 6-well plates.

Parameter recording

[00166] Cell viability was assessed 24 hours after thawing or post-passage (for non-vitrified controls) in detached cells after trypsinization using a trypan blue exclusion assay (TC20 Cell Counter, Bio-Rad).

[00167] Confluence was assessed at the indicated times by analyzing 5 random images per well using the DP22 microscope camera together with the CKX-CCSW Confluency Checker software (Olympus).

Human-induced pluripotent stem cells (hiPSCs) Cell origin and culture

[00168] A hiPSC strain (dKIPs, gift from Prof.

Dr F. Edenhofer, U. Bonn) underwent slow freezing using CDCS. They were grown in vitronectin coated 6-well cell culture plates in Essential 8™ Flex medium (Gibco) supplemented with 0.5% Pen/Strep (Gibco) at 37°C with 5% CO 2 .

Slow freeze (SLF)

[00169] For SLF, culture plates were washed gently with D-PBS, cells were separated as aggregates from wells using ReLeSR™ (StemCell Technologies), suspended in Essential 8™ -Flex medium, pooled and evenly distributed into three groups . One group received no treatment and was placed back in culture directly (non-cryopreserved control). After centrifugation, cell pellets were resuspended in CryoStor® CS10 (STEMCELL Technologies) or in a 50% dilution of CDCS in D-PBS at a concentration corresponding to 1 culture well/ml. 1 ml aliquots (equivalent to the amount of cells in 1 well) were then distributed into 1.8 ml cryotubes (Nunc) which were placed overnight in Mr Frosty™ devices (Nalgene) in a -80°C freezer before of storage on LN2. Cells were stored in the LN2 tank for at least 4 h.

thawing

[00170] The frozen cells were later heated by removing the cryotubes from the LN2, bringing and keeping their lower part in a water bath at 37 °C.

Immediately after thawing, 1 ml of Essential 8™ Flex culture medium was added and mixed by gently pipetting up and down. The cell suspensions were then transferred to conical tubes containing 3 ml of culture medium. Each sample was made up to 10 ml by adding culture medium, centrifuged and resuspended in 1 ml of fresh medium. Each sample was seeded into four wells of vitronectin-coated 6-well plates.

Parameter recording

[00171] Cell viability was assessed 24 hours after thawing (or post-passage for non-frozen controls) on detached cells (StemPro® Accutase® Cell Dissociation Reagent, Gibco) using a trypan blue exclusion assay (TC20 Cell Counter , Bio-Rad).

[00172] Confluence was assessed at the indicated times by analyzing 5 random images per well using the DP22 microscope camera together with the CKX-CCSW Confluency Checker software (Olympus).

Results Physical test of CDCS vitrification capacity

[00173] As shown in Table 3, CDCS does not undergo any crystallization after cooling in LN2 nor after heating in a water bath at 37 °C. Under the same standardized conditions, VSE crystallized after cooling in half of the samples, while all samples of CDCS(-) (precursor of CDCS without PVA) crystallized after heating. In conclusion, both the reference VSE solution and CDCS(-) (CDCS without PVA) do not allow consistent vitrification and allow rapid crystal formation on cooling (VSE) or heating (CDCS(-)) in the proposed system with relatively high thermal inertia. This rapid crystallization of VSE and CDCS(-) is expected to be deleterious to the cells it may contain. CDSC is therefore shown to be a strong ice crystal inhibiting solution, even stronger than a renowned vitrification composition (VSE, whose composition is very similar to commercial solutions used in Medical Assisted Breeding (MAP)), and in conditions that cause slower rates of cooling and heating than generally recommended.

Table 3: Physical vitrification tests of VSE, CDCS(-) and

CDCS Straw ID solution Cooling Heating vitrification VSE VSE1 V V VSE2 C C VSE3 C C VSE4 V V CDCS(-) CDCS(-)1 V C CDCS(-)2 V C CDCS(-)3 V C CDCS(-)4 V C CDCS CDCS1 V V CDCS2 V V CDCS3 V V CDCS4 V V This table reports the results of a standardized physical vitrification test. Three vitrification solutions (VSE, CDCS(-) and CDCS) were aspirated and sealed in 250 l of French straw, cooled in LN2 and subsequently heated in a water bath at 37 °C. The vitrified or crystallized states were assessed on cooling and heating by visual examination. “V” stands for vitrification and “C” stands for crystallization.

CDCS stability tests

[00174] CDCS underwent stability testing when stored for several weeks at 4 °C, room temperature, 37 °C and combinations of room temperature or 37 °C with further storage at 4 °C.

As shown in Table 4, and despite the absence of any antimicrobial agent, no noticeable change in CDCS was observed over time, whatever the storage conditions, as assessed by visual inspection of the solution (no turbidity or precipitates) or its effectiveness in the vitrification of embryos.

[00175] In conclusion, the properties of the CDCS composition are remarkably stable over time, even under unfavorable temperature conditions and despite the absence of any antimicrobial agent. In fact, storage at 4 °C as well as 37 °C (and also in intermediate and mixed temperature conditions) for up to 6 months does not change its optical/physical aspect, nor its high efficiency in the vitrification of murine embryos. .

Table 4: CDCS stability tests under various storage conditions Storage at 4 °C No. of No. of % of No. % of Inspection Moment Embryos Survival Survival Blastocysts Visual Blastocysts D1 21 21 100% 14 67% OK D7 21 21 100% 14 67% OK

D14 19 19 100% 19 100% OK M1 20 20 100% 19 95% OK M2 21 21 100% 19 90% OK M3 20 20 100% 18 90% OK M6 20 20 100% 14 70% OK

Room temperature storage No. of No. of % No. of % Inspection Moment Embryos Survival Survival Blastocysts Blastocysts Visual D7 21 21 100% 13 62% OK D14 20 20 100% 12 60% OK M1 21 21 100% 16 76% OK M3 21 21 100% 14 67% OK M6 20 20 100 % 17 85% OK

Storage at room temperature (14D) then 4°C No. of No. of % No. of % Inspection Moment Embryos Survival Survival Blastocysts Blastocysts Visual D14 21 21 100% 13 62% OK M1 21 21 100% 18 86% OK M3 21 21 100% 13 62% OK M6 17 17 100% 14 82% OK

Storage at 37°C No. of No. of % No. of % Inspection Moment Embryos Survival Survival Blastocysts Blastocysts Visual D7 21 21 100% 12 57% OK D14 21 20 95% 14 70% OK M1 20 20 100% 19 95% OK M2 21 21 100% 16 76% OK M3 18 18 100 % 15 83% OK M6 20 20 100% 20 100% OK

Storage at 37°C (14D) then 4°C No. of No. of % No. of % Inspection Moment Embryos Survival Survival Blastocysts Visual Blastocysts D14 21 21 100% 17 81% OK M1 20 20 100% 20 100% OK M3 12 12 100% 11 92% OK M6 21 21 100% 20 95% OK This table shows the results of stability tests performed on aliquots of CDCS under various storage conditions (4 °C, room temperature, room temperature for 14 days followed by storage at 4 °C, 37 °C and 37 °C for 14 days followed by storage at 4°C). The recording times were 7 days (D7), 14 days (D14), 1, 2, 3 or 6 months (M1, M2, M3, M6) after the solution was manufactured. After visual inspection of the aliquots for normal appearance, it was used as a vitrification solution for one-step vitrification of 2-cell stage murine FVB/N embryos.

For each end point, the number of vitrified embryos, their immediate survival after heating, and their development to the blastocyst stage were recorded.

Cryopreservation of embryos, MII oocytes and cells with CDCS Mouse embryos

[00176] Two-cell stage mouse embryos of the C57BL6/JRj strain were subjected to two vitrification/heating protocols using either the reference VSE vitrification solution or undiluted CDCS.

Briefly, the cooling was the same (one step) for both protocols, but the heating was different. Heating was at a fast rate by directly immersing the vitrified drop containing the embryos in warm culture medium or at a slower rate due to a first heating step in open air for 10 seconds before immersion in the medium (see M&M). As shown in Table 5, after slow heating (air), early embryonic survival was better with CDCS than with VSE (p = 0.026).

Table 5: Vitrification and fast/slow heating of mouse embryos with CDCS

At the. of No. of % No. of % Treatment/session Embryos Survival Survival Blastocysts Blastocysts CDCSmed/1 19 13 68% 8 42% CDCSmed/2 36 34 94% 28 78% CDCSmed/Total 55 47 85% 36 65% CDCSair/1 19 18 95% 12 63% CDCSair/2 36 32 89% 22 61% CDCSair/Total 55 50a 91% 34 62% VSEmed/1 20 13 65% 5 25% VSEmed/2 36 32 89% 25 69% VSEmed/Total 56 45 80% 30 54% VSEair/1 19 11 58% 6 32% VSEair/2 37 31 84% 26 70% VSEair/Total 56 42b 75% 32 57% Control/1 16 16 100% 11 69% Control/2 24 24 100% 23 96% Control/Total 40 40 100% 34 85% This table shows the results of vitrification and heating of 2-cell stage mouse embryos

C57BL6/JRj.

Along with non-cryopreserved controls, two separate experiments were conducted (sessions 1 and 2). CDCS was compared to VSE in one-step vitrification protocols followed by heating by direct immersion of the embryo in the culture medium (med) or by slower rate heating in air followed by immersion in the medium (air). The numbers of embryos submitted to the tests are indicated (No. of Embryos) together with those that survived the treatment (No. of Survival),

evolving into the blastocyst stage on D5 (No.

blastocysts). Where relevant, survival and development values were superscripted, compared with each other using a Chi-square test (1 df) and the values of

Corresponding P were calculated: ab: 0.026.

mouse MII oocytes

[00177] Mice of strains C57BL6/JRj and FVB/NRj were subjected to one-step vitrification with undiluted CDCS. Subsequently, they were fertilized in vitro with frozen sperm and allowed to develop in vitro to the blastocyst stage or transferred to pseudopregnant mothers to monitor their development in vivo (parameter = live birth).

As shown in Table 6, vitrification of oocytes with CDCS allows efficient in vitro fertilization as well as in vitro and in vivo development of the resulting embryos at rates similar to those observed with usual vitrification protocols (F.J. Ectors, personal communication).

Table 6: Development of embryos produced after IVF from oocytes vitrified with CDCS Treatment/ Oocytes Zygotes 2 cells Blastocysts Newborns No. of No. of Cepa No. At the. % At the. % At the. % At the. % start transf. C57BL6/JRj 382 362a 95% 191c 50% 105 85e 81% 50 29g 58% NV control 466 447b 96% 310d 67% 132 102f 77% 65 32h 49% FVB/NRj 414 337i 81% 230k 29o 36% NV control 514 501j 97% 400l 78% 185 163n 88% 65 31p 47% This table shows the results of C57BL6/JRj and FVB/NRj development of embryos produced by IVF from one-step vitrified mouse oocyte with CDCS . Unvitrified controls (NV) were produced in the same way with fresh unvitrified oocytes. The numbers of oocytes subjected to testing are indicated (No. of Oocytes) along with the resulting zygotes (No. of Zygotes) and 2-cell stage embryos (No. of 2 cells). Some 2-cell stage embryos were kept in culture (No. from start) to assess development to the blastocyst stage (No. from Blastocysts), or were transferred (No. from transf.) to assess the number of newborn resulting births (No. of newborns). All corresponding percentages in relation to the initial number of oocytes are indicated. Developmental values after oocyte vitrification were compared to NV controls, and were superscripted, compared to each other using a Chi-square test (1 df) and the corresponding P values were calculated: ab: 0.423; cd: 1.121 x 10-6; ef: 0.490; gh: 0.350; ij: 1.987 x 10-16; kl: 5.153 x 10-13; mn: 1.002 x 10-10; op: 0.164.

human blastocysts

[00178] As shown in Table 7, aliquots of diluted and undiluted CDCS were used in a multi-step procedure to vitrify aneuploid blastocysts. From 20 embryos, 18 and 17 appeared healthy and intact directly after heating and after 18 hours of culture, respectively.

Table 7: Vitrification of human aneuploid blastocysts with CDCS Protocol (D5 human aneuploid blastocysts) CDCS cooling in D-PBS 25 50

(%) Exposure time 4 to 5 4 (min) SUC heating in HTF/HSA (8/2; 1 0.75 0.5 0.25 M) Exposure time 0.5 0.5 1 2 (min) Results (D5 human aneuploid blastocysts) Total number treated Post-warming survival at time t=0 t=18 hours 20 18 intact 17 intact This table shows the cooling protocol applied with CDCS dilutions (top table) and the heating protocol with SUC. CDCS dilutions are expressed as % (v/v) and SUC content as a molarity (M). HTF is a culture medium for embryos, HSA stands for human serum albumin. The survival/integrity of the blastocysts directly after heating and after 18 hours of culture is shown in the lower frame.

human MII oocytes

[00179] As shown in Table 8, aliquots of diluted and undiluted CDCS were used in a multi-step procedure to vitrify human MII oocytes. All 10 oocytes submitted to the study appeared healthy and intact shortly after heating and after 18 hours of culture, respectively.

Table 8: Vitrification of human MII oocytes with CDCS Protocol (human MII oocytes) Cooling

CDCS in D-PBS 6.25 12.5 25 50 100 (%) Exposure time 3 3 3 5 1 (min) Heating SUC in HTF/HSA (8/2; 1 0.5 0.25 0.125 M) Time exposure 1 3 2 2 (min) Results (human MII oocytes) Total number treated Post-warm survival at time t=0 t=18 hours 10 10 intact 10 intact This table shows the cooling protocol applied with CDCS dilutions (table upper) and the heating protocol with SUC. CDCS dilutions are expressed as % (v/v) and SUC content as a molarity (M). HTF is a culture medium for embryos, HSA stands for human serum albumin. Oocyte survival/integrity directly after heating and after 18 hours of culture is shown in the lower frame.

Equine mesenchymal stem cells One-step vitrification and heating

[00180] One-step vitrification followed by one-step heating procedures were performed on EC-MSCs using CDCS in cryotubes. Twenty aliquots of 106 cells were subjected to the vitrification/warming procedure along with 17 unvitrified controls. Cell viability and confluence values (which is a composite indicator of viability and proliferation capacity) of vitrified samples were very similar to controls (Figure 1), although the viabilities were statistically different, probably due to the high repeatability of the results (SD reduced) and inevitable cell loss during the handling process itself. Furthermore, the morphological aspects of EC-MSC cultures after one-step vitrification using CDCS or as unvitrified controls are indistinguishable (Figure 2).

Slow freezing and thawing

[00181] SLF was performed on EC-MSCs using 2x and 4x diluted CDCS as well as a commonly used reference method (10% DMSO, v/v). Fig. 3 shows that both CDCS dilutions are as efficient as the reference method, ending up with cell viability almost equal to that of non-cryopreserved cells 24 hours after heating. The same applies for cell confluence where all three treatments give similar results, exhibiting a confluence delay of approximately 1 day compared to non-cryopreserved cells.

Human-induced pluripotent stem cells (hiPSCs)

[00182] SLF was performed on hiPSCs using 2x diluted CDCS and an optimized commercial solution (CryoStor® CS10). As shown in Fig. 4, cell viability using diluted CDCS averages 29.6% 24 hours after thawing, which is the same order after using CryoStor CS10 (41.8%), although statistically different. Furthermore, confluence remains similar over time (1 to 7 days), reaching the level of non-cryopreserved cells on day 5.

[00183] The results provided in this document demonstrate that the compositions of the present invention provide efficient, stable, biologically safe and reproducible solutions for cryopreserving live cells, embryos and gametes from various organisms, including humans, following various methods, using various devices and in different ways. diluted or undiluted.

Example 2: Compositions of the present invention (CDCS) enable more efficient cryopreservation of murine embryos than Ref1 and Ref2 solutions Materials and Methods Cryopreservation Solution Compositions

[00184] The Chemically Defined Cryopreservation Solution (CDCS) is composed as indicated in table 1 for its reference stock concentration (1x concentrate). According to protocols, this core solution can be used undiluted or diluted to meet unique methodological/cryobiological requirements. The amounts of each of the CDCS components in the undiluted stock may vary relatively from one another within the ranges indicated in Table 1, without significantly altering the conditioning or cryoprotection properties.

[00185] Reference cryopreservation solution 1 (also referred to herein as "Ref1 solution") refers to a cryopreservation solution as described in international patent application WO 2010/046949 A1 having a composition as described in table 9.

Table 9: Composition of the Ref1 solution Component Concentration Ethylene glycol 7.5% DMSO 7.5% Sucrose 0.5M PVA 0.1% Ficoll 1% Hyaluronan 0.05% Buffered culture solution in

HEPES

[00186] Reference Cryopreservation Solution 2 (also referred to in this document as "Ref2 solution") refers to a solution described in "Lopez M. et al, Chemically defined and xeno-free cryopreservation of human adiose-derived stem cells, 2016 , PLOS ONE, vol. 11 (3): 1 to 15" having a composition as described in table 10.

Table 10: Composition of the Ref2 solution Component Concentration Ethylene glycol 3.5% DMSO 3.5% HEPES buffered culture medium containing 0.25M Trehalose PVA 2%

Ficoll 5% EGTA 0.1mM Physical test of vitrification capacity

[00187] The vitrification capabilities of CDCS, Ref1 solution and Ref2 solution (ie, their solidification without crystal formation) after cooling and the absence or formation of crystal after subsequent heating were evaluated using a physical test. CDCS, Ref1 solution or Ref2 solution were aspirated with a syringe into 250 µl French straws (Minitube ref. 13407/0010) which were subsequently closed with a plug at the lower end. The straws were dipped and shaken in liquid nitrogen (LN2) and then heated in a water bath at 37 °C.

mouse embryos

[00188] FVB/N x CD1 mice were used in this example. Fertilized oocytes were provided by the Central Mouse Facility at the University of Liège. Zygotes with two pronuclei and normal-appearing cytoplasm were kept for later use. If applicable, they were cultured in drops of 50 l of M16 medium under mineral oil, in a saturated atmosphere of water at 37 °C under 5% CO2 until treatment.

Embryos in the blastocyst stage (~5 days of development) were submitted to the vitrification/warming process.

Glazing and heating

[00189] Embryo vitrification was performed using a one-step method using the CDCS described above, Ref1 solution solutions or Ref2 solution (hereinafter referred to as "solutions").

Preloading the straws

[00190] 0.25 ml French straws (CryoBioSystem) were used as carriers to vitrify the embryos.

[00191] The straw is connected from the cotton plug side to a 1 ml syringe. Fifty millimeters (mm) of DPBS (Gibco) supplemented with 10% fetal bovine serum (Gibco) (hereinafter referred to as DPBS-F10) is loaded, followed by aspiration of 10 mm of air. The filled straws are arranged horizontally at room temperature until use (embryo loading).

Conditioning and cooling of embryos

[00192] Briefly, groups of a maximum of 5 embryos are harvested from their culture medium and transferred to a drop of 500 l solution. They are moved several times into the solution to eliminate their culture medium gangue. Then they are harvested and transferred to 50 µl drops of solution. Embryos are suction loaded into the straw along with a 10 mm column of solution, followed by 10 mm of air and finally by DPBS-F10 until the cotton is wetted with the pre-filled DPBS-F10 column.

loaded. The straw is sealed and dipped directly and shaken in liquid nitrogen (LN2). From the transfer of the embryos to the solutions to their immersion in LN2, it takes no more than 60 seconds.

Embryo heating and cultivation

[00193] Heating was performed at least 24 hours after cooling. The straw is removed from the LN2, allowed to warm for 5 seconds in air and directly immersed in a water bath at 37 °C for another 5 seconds. The straw is cleaned and cut with scissors at the sealed end. The straw contents are expelled in a drop of 800 µl DPBS-F10 at room temperature with a metal rod pushing the cotton plug. Embryos are rinsed, harvested and transferred for final wash in another 200 µl DPBS-F10 drop. After rinsing, the embryos are transferred to a 50 l drop of M16 medium under mineral oil, in a saturated atmosphere of water at 37 °C under 5% CO2 until further analysis.

Parameter recording

[00194] For the physical test of vitrification potential, the vitrification/crystallization state of the solutions after cooling and subsequent heating was assessed by visual examination: a vitrified solution remains clear and transparent while crystallization induces a white/milky appearance.

[00195] For the embryo vitrification assays, the parameters recorded were the survival in culture 24 and 48 hours after heating and the hatching of the zona pellucida in 48 hours. Survival was evaluated by evaluating zona pellucida and blastomeres morphology.

Results Comparison of solution vitrification capabilities

[00196] As shown in Table 11, CDCS does not undergo any crystallization after cooling in LN2 nor after heating in a water bath at 37 °C. Under the same standardized conditions, the Ref1 solution and the Ref2 solution undergo crystallization during cooling and heating. In conclusion, both the Ref1 and the Ref2 solutions do not allow consistent vitrification and allow rapid crystal formation on cooling and heating in the proposed system with relatively high thermal inertia. This rapid crystallization of the Ref1 solution and the Ref2 solution is expected to be deleterious to the cells it may contain. CDSC, therefore, is shown to be a strong ice crystal inhibiting solution under conditions that cause slower rates of cooling and heating (~1000 °C/min) than generally recommended (~20,000 °C/min).

Table 11: Physical vitrification tests of Ref1 solution and Ref2 solution and CDCS Straw ID solution Cooling Heating cryopreservation Ref1 Solution Ref1-1 C Ref1-2 C C Ref1-3 C C Ref1-4 C C Ref2 Solution Ref2-1 C C Ref2-2 C C Ref2-3 C C Ref2-4 C C CDCS CDCS1 V V CDCS2 V V CDCS3 V V CDCS4 V V This table reports the results of a standardized physical vitrification test. Three vitrification solutions (Ref1 solution, Ref2 solution and CDCS) were aspirated and sealed in 250 l of French straw, cooled in LN2 and subsequently heated in a water bath at 37 °C. The vitrified or crystallized states were assessed on cooling and heating by visual examination. “V” stands for vitrification and “C” stands for crystallization.

Embryo vitrification assays

[00197] It seems obvious from the results reported in Table 12 that, using a one-step vitrification procedure in a system with a relatively high thermal inertia, CDCS guarantees much better survival and hatch rate than Ref1 and Ref2.

Table 12: Vitrification and rapid rate heating of mouse embryos with Ref1 solution, Ref2 solution and

CDC

24H post-warm analysis 48H post-heat analysis No. of No. of % of No. of % of No. of % Treatment Embryo Surv.

Survival

Survival

Survival hatched hatched CDCS 143 75a 52% 71d 50% 12g 8% Solution 137 2b 1% 0e 0% 0h 0% Ref1 Solution 140 2c 1% 0f 0% 0i 0% Ref2 This table shows the results of vitrification and heating of embryos in mouse blastocyst stage

FVB/NxCD1 (D5), performed together with non-cryopreserved controls.

CDCS was compared with the Ref1 solution and the Ref2 solution in one-step vitrification protocols,

followed by heating by direct expulsion of the embryo into the culture medium.

The numbers of embryos submitted to the tests are indicated (No. of Embryos) together with those that survived the treatment 24 hours and 48 hours post-warming (No.

Survival), evolving to the hatched blastocyst stage 48H post-warm (hatched No.). Values with different superscripts were compared to each other and the corresponding P values were calculated using chi-square tests:

a-b: 1.3 x 10-21; a-c: 5.3 x 10-22; d-e: 1.3 x 10-21; d-f: 5.8 x

10-22; g-h: 5.3 x 10-4; g-i: 4.6 x 10-4.

Claims (15)

1. Liquid composition for cryopreservation of a biological material, characterized in that it comprises - a polar aprotic solvent selected from dimethylsulfoxide (DMSO) and dimethylformamide (DMF), - a monohydric or polyhydric alcohol selected from ethylene glycol, propylene glycol, glycerol , erythritol, sorbitol, mannitol, xylitol, volemitol, methanol, ethanol, isopropanol, butanol and pentanol; - an unbranched polysaccharide selected from the group consisting of sucrose, trehalose, lactulose, melibiose, lactobionate, raffinose and cellulose, preferably sucrose; - dextran; and - polyvinyl alcohol. 2. Liquid composition for cryopreservation of a biological material, according to claim 1, characterized in that it comprises DMSO, ethylene glycol, sucrose, dextran and polyvinyl alcohol. 3. Liquid composition according to claim 2, characterized in that the composition comprises substantially equal amounts of DMSO and ethylene glycol and a sucrose concentration that is at least 8 times that of dextran. 4. Liquid composition, according to any one of claims 1 to 3, characterized in that it comprises - at least 0.8% (v/v) of a polar aprotic solvent; - at least 0.8% (v/v) of a monohydric or polyhydric alcohol; - at least 0.5% (w/v) of unbranched polysaccharide; - at least 0.037% (w/v) of dextran; - at least 0.015% (w/v) of polyvinyl alcohol; and - up to 100% (v/v) of a diluent, preferably with pH 7.2 to 7.4. 5. Liquid composition, according to any one of claims 1 to 4, characterized in that it comprises: - 13.0 to 23.0% (v/v), preferably 17.7% (v/v) of a polar aprotic solvent; - 13.0 to 23.0% (v/v), preferably 17.7% (v/v), of a monohydric or polyhydric alcohol; - 9.0 to 31.0% (w/v), preferably 22.7% (w/v), of an unbranched polysaccharide; - 0.60 to 22.0% (w/v), preferably 2.64% (w/v), of dextran; - 0.30 to 4.0% (w/v), preferably 0.88% (w/v), polyvinyl alcohol; and/or - up to 100% (v/v) of a diluent, preferably with pH 7.2 to 7.4; or a dilution of said liquid composition in said diluent. 6. Liquid composition, according to any one of claims 1 to 5, characterized in that it comprises at least 15% (v/v) of a polar aprotic solvent, at least 15% (v/v) of a monohydric alcohol or polyhydric, at least 15% (w/v) of an unbranched polysaccharide, at least 2% (w/v) of dextran and about 0.9% (w/v) of polyvinyl alcohol. 7. Liquid composition according to any one of claims 4 to 6, characterized in that said diluent is a balanced saline solution, preferably phosphate buffered saline (PBS). 8. Liquid composition, according to any one of claims 2 to 7, characterized in that dextran comprises one or more of Dextran 1, Dextran 40 and/or Dextran 70. 9. Liquid composition, according to any one of claims 1 to 8, characterized in that it comprises - 17.7% (v/v) of DMSO; - 17.7% (v/v) ethylene glycol; - 22.7% (w/v) sucrose; - 0.88% (w/v) Dextran 1; - 0.88% (w/v) Dextran 40; - 0.88% (w/v) Dextran 70; and - 0.88% (w/v) of polyvinyl alcohol. 10. Liquid composition, according to any one of claims 1 to 9, characterized in that the liquid composition does not comprise proteins, polypeptides or peptides. 11. Use of the liquid composition as defined in any one of claims 1 to 10 characterized in that it is for cryopreservation of a biological material, wherein freezing is preferably carried out by a freezing procedure, a multiple vitrification procedure steps or a one-step glazing procedure. 12. Method to preserve a biological material, characterized by the fact that it comprises the steps of - providing a biological material; - contacting the biological material with the liquid composition as defined in any one of claims 1 to 10; - cooling and/or heating the biological material in the liquid composition as defined in any one of claims 1 to 10. 13. Method according to claim 12, characterized in that it comprises contacting the biological material with at least one dilution of a composition according to any one of claims 5, 6 or 9, or a claim dependent thereon, before contacting the material biological product with said composition. 14. Method according to any one of claims 12 or 13, characterized in that said cooling comprises freezing the biological material or vitrifying the biological material, preferably a vitrification in one or several stages of the biological material . 15. Use, according to claim 11, or method, according to any one of claims 12 to 14, characterized in that the biological material is selected from the group of stem cells, gametes and embryos.